The invention relates to a matrix in ball form, a method for preparing such a matrix and the use thereof as a cell carrier.
The invention applies to the field of cell culture, and in particular the expansion of cells used in the context of cell therapy, in bioproduction to produce bioactive molecules, or in research and development as cell models. Such cells are for example primary cells, pluripotent stem cells, stem cell lines or immortalized cell lines.
In particular, cell therapy, based on the use of adult stem cells, consists of replacing and regenerating the cells of unhealthy tissue by administering healthy and functional cells that have been selected, amplified and/or modified ex vivo.
The cell expansion step is necessary, since the different stem cell sources do not make it possible to obtain a sufficient quantity of cells for clinical use.
Currently, the adherent stem cells are cultivated in two-dimensional systems, in particular in plastic culture containers.
To obtain a larger quantity of cells, it is necessary to use a larger culture surface, for example by using a larger number of culture containers, increasing the size of the culture containers or using multilevel culture containers such as CellStack™ (Corning, USA). In these cases, however, the handling of the culture containers becomes more laborious.
Three-dimensional culture systems have also been tested, in particular using microcarriers in suspension.
For example, document US 2012/0009645 proposes a method for cultivating the pluripotent stem cells (embryonic stem cells, induced pluripotent stem cells) on microbeads, in the presence of a ROCK inhibitor. The microbeads are for example D53 cellulose beads or Cytodex 1 or 3 beads.
However, the use of such synthetic microcarriers has several drawbacks. First of all, these microcarriers have a low cell inoculation rate (less than 30%), causing a massive loss of cells of interest from the beginning of the expansion process. Next, the detachment of the cells from their carrier and the carrier-cell separation before injection into the patient remains a sizable problem. Lastly, when these synthetic microcarriers are used, they release plastic particles that cannot be blocked and that may therefore be injected into the patient at the same time as the cells of interest.
To offset the problem of cell adherence, it has been proposed to coat the microcarriers with molecules. However, these molecules are often expensive and/or of animal origin, which raises a biological safety issue.
For example, document US 2011/0111498 proposes a method for cultivating stem cells (mesenchymal stem cells, embryonic stem cells or induced pluripotent stem cells) on microcarriers in suspension. The microcarriers are for example synthetic beads (Toyopearl, Cytodex, DE52) having a positive charge and a coating of the Matrigel™, laminin or hyaluronic acid type capable of supporting cell growth.
Document WO 2009/134197 also describes a microcarrier, such as a Cytodex, for cell culture, coated with a plant protein hydrolysate, such as a soy peptone hydrolysate. This microcarrier has the advantage of not comprising animal proteins.
Document WO 2011/017050 proposes a microcarrier with no proteins of animal origin for cell culture having a polymer coating (e.g., swelling methacrylate) on which a polypeptide is grafted comprising the RGD sequence to promote attachment of the cells.
Cell microcarriers based on natural components also exist.
For example, document EP 1 015 570 proposes fibrin(ogen) microbeads as a cell carrier. These microbeads are prepared from fibrinogen, thrombin and factor XIII. However, the thrombin used as cross-linking agent is exogenous, which increases the preparation difficulty and the biological risk.
In the document by Eibes G., et al. (Maximizing the ex vivo expansion of human mesenchymal stem cells using a microcarrier-based stirred culture system. Journal of biotechnology, 2010, vol. 146, no. 4, pp. 194-197), it was shown that gelatin microporous beads (Cultispher S) are capable of adhering and causing mesenchymal stem cells to proliferate.